The ubiquitin-mediated proteolysis system is the major pathway for the selective, controlled degradation of intracellular proteins in eukaryotic cells. Ubiquitin modification of a variety of protein targets within the cell appears to be important in a number of basic cellular functions such as regulation of gene expression, regulation of the cell-cycle, modification of cell surface receptors, biogenesis of ribosomes, and DNA repair. One major function of the ubiquitin-mediated system is to control the half-lives of cellular proteins. The half-life of different proteins can range from a few minutes to several days, and can vary considerably depending on the cell-type, nutritional and environmental conditions, as well as the stage of the cell-cycle.
Targeted proteins undergoing selective degradation, presumably through the actions of a ubiquitin-dependent proteosome, are covalently tagged with ubiquitin through the formation of an isopeptide bond between the C-terminal glycyl residue of ubiquitin and a specific lysyl residue in the substrate protein. This process is catalyzed by a ubiquitin-activating enzyme (E1) and a ubiquitin-conjugating enzyme (E2), and in some instances may also require auxiliary substrate recognition proteins (E3s). Following the linkage of the first ubiquitin chain, additional molecules of ubiquitin may be attached to lysine side chains of the previously conjugated moiety to form branched multi-ubiquitin chains.
The conjugation of ubiquitin to protein substrates is a multi-step process. Ubiquitin is a small, highly curved protein which must be activated before it is transferred to a substrate protein. Accordingly, in an initial ATP requiring step, a thioester is formed between the C-terminus of ubiquitin and an internal cysteine residue of a ubiquitin activating enzyme, E1. Activated ubiquitin is then transesterified to a specific cysteine on one of several E2 enzymes. Finally, these E2 enzymes transfer ubiquitin to a lysine residue of a protein substrate. Substrates are recognized either directly by ubiquitin-conjugated enzymes or by associated substrate recognition proteins, the E3 proteins, also known as ubiquitin ligases. A major cellular mechanism by which proteins are degraded in eukaryotic cells is by ubiquitinylation of the protein, thereby targeting the protein for degradation by the 26S proteasome (Hochstrasser (1995) Curr. Opin. Cell Biol. 7:215). Ubiquitin is a small, highly conserved protein, which must be activated before it is transferred to a substrate protein. Activation of ubiquitin occurs through formation of a thioester bond between the COOH terminus of the ubiquitin molecule and a ubiquitin-activating enzyme, E1. Ubiquitin is then transesterified to one member of a family of a ubiquitin conjugating enzymes, E2 enzymes. Ubiquitin is then transferred, either directly or indirectly, to a lysine residue of a substrate protein. Transfer to the substrate protein may require the assistance of a ubiquitin ligase also termed E3 enzyme or complex. An E3 is generally required for the formation of multiubiquitin chains on the substrate, a step that facilitates efficient recognition of the substrate by the proteosome. It has been suggested that E3 is the primary source of substrate specificity in the ubiquitination cascade, as some E3s have been shown to directly bind substrates (Hershko et al. (1986) J. Biol. Chem. 261:11992; Bartel et al. (1990) EMBO J. 9:3179). Furthermore, in some situations, a ubiquitin molecule is first transferred from an ubiquitin conjugating enzyme to an E3 enzyme or complex, prior to being transferred to the substrate protein (Willems et al., supra).
Ubiquitination of proteins and subsequent protein degradation plays an important role in various steps of the cell cycle and is thus crucial in the regulation of cell proliferation and differentiation. Briefly, cell-cycle events are thought to be regulated by a series of interdependent biochemical steps. In eukaryotic cells mitosis does not normally take place until the G1, S and G2 phases of the cell-cycle are completed. In all eukaryotic cells examined to date, the cell cycle appears to be regulated by the sequential activation of a series of the CDK""s or Cyclin Dependent Kinases (reviewed in Morgan, (1995) Nature 374:131-134; King et al., (1994) Cell 79:563-571; Norbury and Nurse, (1992) Annu. Rev. Biochem. 61:441-470). Yeast cells contain a single CDK known as cdc2 in S. pombe (Beach et al., (1982) Nature 300:706-709; Booher and Beach, (1986) Gene 31:129-134; Hindley and Phear, (1984) Gene 21:129-134; Nurse and Bissett, (1981) Nature 292:558-560; Simanis and Nurse, (1986) Cell 45:261-268; and for review see Forsburg and Nurse, (1991b) Annu. Rev. Cell Biol. 7:227-256) and cdc28 in S. cerevisiae. Drosophila and vertebrates have several CDKs, including CDK1, CDK2, CDK4, and CDK6 (Elledge S. J. (1996) Science 274:1664).
The activity of the CDKs is controlled at least in part by the association of the CDKs with various cyclins during progression through the cell cycle. Cyclins also contribute to substrate specificity The CDK-cyclin complex is both positively and negatively regulated by several mechanisms including phosphorylation, binding to inhibitors (CKIs) and other proteins such as Suc1 (Cks1) that might modify their specificity or accessibility to regulators, and protein degradation by the ubiquitin conjugation pathway (Patra et al. (1996) Genes Dev. 10:1503).
In addition to their role in activating mitosis, the cyclin-CDKs are required to stimulate the initiation of DNA replication. In yeast, the activity of cyclin-CDKs is blocked specifically by the inhibitor Sic1, which is present in cells from late mitosis until shortly after START. Thus, degradation of Sic1 is necessary for DNA replication. In yeast, it has been shown that degradation of this protein is mediated by the ubiquitin conjugating enzyme (E2) Cdc34 and also requires cdc4 and cdc53 and a Skp1 protein. These proteins are also involved in degradation of other cell cycle regulatory proteins in yeast, including the G1 cyclins, which are required for executing the START of the cell cycle (King et al. (1996) Science 274:1653; Willens et al. (1996) Cell 86:453; Bai et al. (1996) Cell 86: 263; and Mathias et al (1996) Mol. Cell. Biol. 16:6634).
In particular, molecular cloning of cdc34, a gene required for the G1-S transition in budding yeast, revealed that a ubiquitin conjugation step was required just before the initiation of DNA replication. cdc34 encodes a ubiquitin conjugating enzyme that participates in the destruction of multiple proteins, including the G1 cyclins CLN2 and CLN3, as well as proteins not directly related to cell cycle control. However, accumulation of these substrates does not account for the cell cycle arrest of cdc34ts mutants. The nature of the crucial target of cdc34 at the G1-S transition was first implied by genetic studies. A strain deficient in all S-phase and mitotic cyclins recapitulated the cdc34ts mutant phenotype, suggesting that the cdc34 pathway might be required for generating S-phase CDK activity. Extracts made from cdc34ts mutants inhibit S-phase CDKs, implying that cdc34 may be required for the degradation of a CDK inhibitor. A candidate for this activity was SIC1, a tight-binding S-phase CDK inhibitor (Mendenhall (1993) Science 259:216; Nugroho et al. (1994) Mol Cell. Biol. 14:3320). SIC1 is normally degraded as wild-type cells enter S phase, but accumulates in cdc34ts mutants. SIC1 appears to be the crucial substrate blocking progression from G1 to S phase in cdc34ts mutants, because cdc34ts sic1xcex94 double mutants initiate DNA replication at the nonpermissive temperature (Schwob et al. (1994) Cell 79:233). As predicted by these findings, expression of a non-degradable form of SIC1 in wild-type strains blocks cell division at the G1-S transition (King et al. (1996) Science 274: 1652). Ubiquitin-dependent proteolysis of a CDK inhibitor is therefore a crucial mechanism by which the onset of S phase is controlled.
Besides cdc34, three other genes are required for the G1-S transition in budding yeast: cdc4, cdc53, and SKP1 (King et al. supra). Cells with temperature-sensitive mutations in any of these genes exhibit phenotypes similar to that of cdc34ts mutants, and in each case deletion of SIC1 enables these mutants to replicate their DNA. Both cdc53 and SKP1 are members of conserved, multigene families, but there is little information about their biochemical functions. cdc4 contains two recognizable sequence motifs that are found in many unrelated proteins: an F box, which serves as an interaction domain for SKP1 (Bai et al. (1996) Cell 86:263), and eight WD-40 repeats (Neer et al. (1994) Nature 371:297), which may serve as a platform for protein-protein interaction (Sondek et al. (1996) Nature 379:369). Insect cell lysates expressing cdc53, cdc4, and SKP1 (and supplemented with cdc34, ubiquitin, and E1) can sustain ubiquitination of SIC1, suggesting that one of these components functions as an E3 (King et al. supra).
One aspect of the present invention relates to a new class of ubiquitin ligases, the xe2x80x9cSIP ligasesxe2x80x9d for SKP Interacting Proteins. The mammalian homolog of cdc4 is an archetype for the ligase family. The present invention provides isolated and/or recombinant forms of the SIP ligase, and portions thereof. For instance, there is provided isolated and/or recombinant cdc4 polypeptides having an amino acid sequence identical or homologous (e.g. at least 65, 75, 85 or 95%) to the amino acid sequence designated by SEQ ID NO: 2 or 4. The cdc4 polypeptide can have an amino acid sequence encoded by a nucleic acid which hybridizes under stringent conditions to the nucleotide sequence set forth in SEQ ID NO: 1 or 3. The SIP polypeptides of the present invention are preferably encoded by a vertebrate gene, more preferably a mammalian gene, and even more preferably a human gene.
In preferred embodiments, the SIP polypeptides can be components of a ubiquitin ligase complex, e.g., which catalyze ubiquitinylation of a cell cycle regulatory protein such as p27kip1. For instance, the polypeptide is capable of interacting with at least one other protein selected from the group consisting of a component of a ubiquitin ligase, a skp1 protein, a ubiquitin conjugating enzyme, a cullins, and a p27 protein or a (G1 phase) cyclin.
Still another aspect of the present invention provides nucleic acids which encode the subject SIP polypeptides, e.g., which nucleic acid hybridize under stringent conditions to a nucleic acid probe having a nucleotide sequence represented by at least 20, 40, 60, 80 or 100 consecutive nucleotides of SEQ ID NO: 1 or 3, or a sequence complementary thereto. In a preferred embodiment, the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 1 or 3.
The subject nucleic acid can be used to generate expression constructs, such as by placing a transcriptional regulatory sequence in operable linkage with the SIP coding sequence. Accordingly, expression vectors encoding the subject polypeptides can be generated using expression vectors capable of replicating in at least one of a prokaryotic cell and a eukaryotic cell.
Thus, another aspect of the present invention pertains to a host cell transfected with such an expression vector, e.g., expressing recombinant SIP polypeptides, as well as methods of producing a recombinant SIP polypeptide by culturing the instant cell to express the recombinant polypeptide.
The present invention also relates to transgenic animals having cells which harbor a transgene encoding a recombinant SIP polypeptide, or in which the endogenous gene has been inactivated, e.g., by homologous recombination.
Still another embodiment of the present invention relates to isolated nucleic acid which selectively hybridizes under high stringency conditions to at least ten nucleotides of a nucleic acid sequence represented by one of SEQ ID NO: 1 or SEQ ID NO: 3, or complementary sequences thereof, which nucleic acid can specifically detect or amplify a nucleic acid sequence of an vertebrate cdc4. Such nucleic acid can be used, e.g., to generate the expression constructs described above, as well as various assays for detecting SIP genes or transcripts, or for antisense therapy. In a preferred embodiment, the nucleic acid is labeled.
Yet another aspect of the present invention provides reconstituted protein mixtures including a SIP ligase, along with a substrate protein, such as a p27kip1 polypeptide or other CKI protein. The mixture may further include ubiquitin, an E1 enzyme, an E2 enzyme and/or a cullins protein. As appropriate, the E1, E2 or SIP enzymes used to charge the mixture can be provided as transiently ubiquitinated intermediates.
Still another aspect of the present invention pertains to an assay for identifying an inhibitor of SIP-mediated ubiquitination. In a preferred embodiment, the assay comprises a ubiquitin-conjugating system including the substrate polypeptide, ubiquitin and a SIP ligase, under conditions which promote ubiquitination of the substrate polypeptide by the SIP ligase. The ubiquitin-conjugating system is contacted with a candidate agent, and the level of ubiquitination of the substrate polypeptide in the presence of the candidate agent is measured and compared with the level of ubiquitination of the substrate polypeptide in the absence of the candidate agent. A statistically significant decrease in ubiquitination of the substrate polypeptide in the presence of the candidate agent is indicative of an inhibitor of SIP-mediated ubiquitination.
The ubiquitin-conjugating system can be, e.g., a reconstituted protein mixture, a cell lysate or a whole cell. The ubiquitin-conjugating system can also include an E2 ubiquitin conjugating enzyme and/or a cullins protein. The ubiquitin can be provided in such form as (i) an unconjugated ubiquitin, in which case the ubiquitin-conjugating system further comprises an E1 ubiquitin-activating enzyme (E1), an E2 ubiquitin-conjugating enzyme (E2), and adenosine triphosphate; (ii) an activated E1: ubiquitin complex, in which case the ubiquitin-conjugating system further comprises an E2; (iii) an activated E2: ubiquitin complex; and/or (iv) an activated ubiquitin complex with the SIP ligase.
In preferred embodiments, the substrate polypeptide comprises a ubiquitination sequence of a CKI protein, e.g., a CIP/KIP protein such as p27kip1. Likewise, preferred embodiments of the subject assay utilize cdc4 as the SIP ligase, e.g., a vertebrate cdc4, more preferably a mammalian cdc4, and even more preferably a human cdc4 ligase (such as shown in SEQ ID NO: 2).
In certain embodiments of the subject assay, at least one of the ubiquitin and the substrate polypeptide includes a detectable label, and the level of ubiquitination of the substrate polypeptide is quantified by detecting the label in at least one of the substrate polypeptide, the ubiquitin, and ubiquitin-conjugated substrate polypeptide. For illustrative purposes, the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. In one embodiment, the detectable label includes a polypeptide having a measurable activity, e.g., an enzymatic activity, and the substrate polypeptide is fusion protein including the detectable label.
In other embodiments, the amount of ubiquitination of the substrate polypeptide is quantified by an immunoassay, chromatography and/or electrophoresis.
In still other embodiments, the ubiquitin-conjugating system is a host cell expressing the substrate polypeptide and SIP ligase, preferably one of the two being recombinantly produced by the cell.
In yet other embodiments of the subject assay, the reaction mixture is generated to provide a competitive binding assay, e.g., between the test agent and formation of complexes including the subject SIP polypeptides. For example, the assay can be provided as a reaction system including a substrate polypeptide and a SIP polypeptide, under wherein the substrate polypeptide and the SIP polypeptide interact. The mixture is contacted with a candidate agent, and the formation of complexes containing the substrate polypeptide and the SIP polypeptide are measured. A statistically significant decrease in the formation of complexes in the presence of the candidate agent, relative to its absence, is indicative of an inhibitor of the interaction of the substrate polypeptide and the SIP polypeptide. For such assays, the SIP polypeptide can be mutated to lack an endogenous ubiquitination activity, yet retain its ability to bind to the substrate protein.
As above, the competitive screen can be carried out as a reconstituted protein mixture, a cell lysate and/or a whole cell. In the instance of the latter, one embodiment of the subject binding assay provides the substrate and SIP polypeptides as fusion proteins in an interaction trap system.
In any embodiment of the subject assays, one or more of the compounds identified as inhibitors of the SIP-mediated ubiquitination can be formulated as a pharmaceutical preparation, e.g., for further in vivo testing and therapeutic use.
Yet another aspect of the present invention relates to diagnostic assays for determining, in the context of cells isolated from a patient, the level of a SIP transcript, SIP protein and/or SIP ligase activity, which level can be a useful diagnostic/prognostic marker for risk assessment and phenotyping cell and tissue samples. As described herein, the subject assay provides a method for determining if an animal is at risk for a disorder characterized by aberrant cell proliferation, differentiation and/or apoptosis, and also may be used for prognostic purposes when such aberrant cell phenotypes are known.
The subject method can be used for diagnosing a hyperproliferative disorder in a patient which disorder is associated with the destabilization of a CKI protein, such as p27kip1, in cells of the patient, comprising: (i) ascertaining the level of a SIP transcript, SIP protein and/or SIP ligase activity in a sample of cells from the patient; and (ii) diagnosing the presence or absence of a hyperproliferative disorder utilizing, at least in part, the observation of upregulation of a SIP ligase, wherein an increased level of SIP expression or ligase activity in the sample, relative to a normal control sample of cells, can correlate with the presence of a hyperproliferative disorder. In another embodiment, the subject method is a prognostic method for evaluating a cancer patient""s risk of death and/or recurrence of a cancer, comprising (i) ascertaining the level of expression or enyzmatic activity of a SIP ligase in a sample of cancer cells from the patient; and (ii) predicting the patient""s risk of death and/or recurrence of a cancer utilizing, at least in part, that observation, wherein an increased level of expression or activation of the SIP ligase in the sample, relative to a normal control sample of cells, may correlate with an increased risk of death and/or recurrence of a cancer.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press:1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).